We propose to explore the interplay of dynamics and molecular recognition/catalysis in prolyl-tRNA synthetases (ProRSs). These enzymes belong to a family of enzymes known as aminoacyl-tRNA synthetases that are central to protein translation. ProRSs catalyze covalent attachment of proline to tRNAPro and employ inter-domain communication to accomplish their catalytic function. Recent studies revealed that coupled- domain dynamics is prerequisite for the overall function of some bacterial ProRSs. However, the role of these coupled dynamics in molecular recognition/allostery/catalysis is only partially understood. Moreover, ProRSs exhibit significantly diverse domain architecture across species. Yet, very little is known about the species- specific characteristics of intrinsic conformational dynamics (ICD) and their implications towards substrate recognition/discrimination and catalysis. Furthermore, the effect of other cellular macromolecules on ICD has remained unknown for these enzymes. This proposal aims to explore the species-specific ICD and their effects on ProRSs functions using both theory and experiment, and to translate that knowledge into developing a species-specific inhibitor screening protocol. Theoretical and simulation-based studies will generate energetic models for substrate binding and catalysis, which will be calibrated using experiments. If theoretical models are found consistent with experiments, the generated simulation data will be used to extract functional dynamics. Bioinformatics studies will be conducted to correlate evolutionarily constraints with functional dynamics. Finally, a dynamic model of inhibitor screening will be developed by combining electronic and dynamics information. These theoretical results are expected to illustrate key roles of specific amino acids, which could be validated using mutagenesis and kinetic/binding/inhibition studies. Successful completion of the proposed work will enlighten the role of ICD in key functional steps of these modular enzymes. The study will provide deeper insight of the evolutionary pressure that either preserves or optimizes the functional dynamics. In addition, molecular crowding studies are likely to produce valuable details of the perturbation of the energetics-dynamics relationship due to the presence of other macromolecules in a cellular environment. This study will advance the development of novel anti-infective drugs against these promising targets; more potent and species-specific inhibitors of pathogenic AARSs could be designed by taking into account proteins' flexibility and the effect of cellular macromolecules. The proposed work will provide interdisciplinary training for undergraduates. Students will be exposed to advanced concepts of biophysical chemistry and will get hands-on experiences with cutting- edge tools of computational quantum chemistry and bioinformatics, as well as biochemical and molecular biology techniques. A number of talented undergraduate collaborators (9 current students; a total of 40 students) have participated in similar research projects under the supervision of both PIs. Their hard work resulted in 10 peer-reviewed publications since 2009.